Hematopoietic stem cell survival, self-renewal and differentiation is believed to be highly dependent on the stromal microenvironment, which after birth is largely restricted to the bone marrow. However, investigations of candidate bone marrow stromal cells to date have concentrated on fairly ubiquitous cellular elements, such as fibroblasts and endothelial cells. As important as such cells may be in hematopoiesis, their activities cannot easily explain why hematopoiesis concentrates in the marrow. We therefore have recently begun to focus on a specific cell type, the osteoblast, which is located specifically on the endosteal surface within bone marrow cavities. Hematopoietic stem cell (HSC) differentiation occurs in direct proximity to osteoblasts with in the bone marrow cavity Despite this striking affiliation, surprisingly little is known about the precise cellular and molecular impact of osteoblasts on the bone marrow microenvironment. Recently we have shown that human osteoblasts support the growth of primitive human hematopoietic progenitors in long and short term in vitro cultures. To date, no known cytokine alone or in combination can account for this activity. Based upon our preliminary data, osteoblasts appear to directly communicate with primitive hematopoietic cells and support their proliferation. We propose to explore in depth the cellular and molecular basis for osteoblast-stem cell interactions. Specifically, we plan to: 1) Compare the proliferative and self- renewal capacity of primitive hematopoietic cells before and after culture on osteoblast monolayers, utilizing in vitro and in vivo stem cell assays; 2) Determine the effect of bone marrow microenvironmental anatomy and perfusion on osteoblast hematopoietic function; and )3. Molecularly clone osteoblast derived cDNAs whose protein products stimulate the proliferation of primitive human hematopoietic cells. These experiments should give a comprehensive picture of the hematopoietic role of an important but previously uncharacterized cell in the bone marrow microenvironment, a role which we hypothesize to be central to the maintenance and self-renewal of hematopoietic stem cells. Within the context of this Program Project application, these investigations will focus on the role and mechanisms by